Abstract Ribosomal movement along mRNA is an essential facet of protein synthesis in all organisms. During translation elongation the ribosome moves along mRNA in a codon-by-codon manner and unwinds mRNA secondary structure. However, specific mRNA sequences induce ribosome stalling and frameshifting. We aim to understand why these specific RNA sequences slow down ribosome translocation while the ribosome translocates through most mRNA structure without long pauses. Using single-molecule fluorescence microscopy and an in vitro translation system assembled from purified components, we will define properties of frameshift-inducing RNA stem-loops and Shine-Dalgarno-like sequences that are critical for the ribosome pausing. Elucidating the role of RNA structure in the modulation of translation elongation is a key to understanding the molecular mechanism of ribosome frameshifting, which is essential for the synthesis of numerous bacterial, eukaryotic and viral proteins. For example, the production of protease, reverse transcriptase and integrase of human immunodeficiency virus (HIV) depends on a -1 programmed ribosomal frameshifting event. Furthermore, ribosome pausing is a major determinant of numerous co-translational processes including protein processing, folding and targeting to membranes. Modulation of the translation elongation rate also affects mRNA stability and regulates levels of the produced protein. In addition to codon-by-codon translocation, another type of ribosome movement termed ribosome scanning occurs during the initiation phase of protein synthesis in eukaryotes. It is believed that during eukaryotic translation initiation the small ribosomal subunit is first recruited to the 5' end of mRNA and then scans the 5'- untranslated region (5'UTR) of the mRNA for the start codon. The molecular mechanism of scanning, which is critical for correct start codon selection, is poorly understood. Furthermore, the real-time movement of the small ribosomal subunit along the 5'UTR was never detected. We will use a single-molecule fluorescence microscopy assay designed to detect the movement of the small ribosomal subunit and measure the rate of the scanning. We will examine the mechanism of ribosome scanning and gain critical insights into eukaryotic translation initiation, deregulation of which is linked to a number of human diseases including cancer and diabetes. Taken together, the proposed studies will substantially contribute to establishing the molecular mechanisms of protein synthesis and provide a basis for the future development of antiviral and cancer therapies.